The invention relates to the production of transgenic animals.
The field of transgenics has grown rapidly since the initial experiments describing the introduction of foreign DNA into the developing zygote or embryo (Brinster, R. L. et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985), Wagner et al., U.S. Pat. No. 4,873,191 (1989)). Transgenic technology has been applied to both laboratory and domestic species for the study of human diseases (see, e.g., Synder, B. W., et al., Mol. Reprod. and Develop. 40:419-428 (1995)), production of pharmaceuticals in milk (see, for review article, Ebert, K. M. and J. P. Selgrath, xe2x80x9cChanges in Domestic Livestock through Genetic Engineeringxe2x80x9d in Applications in Mammalian Development, Cold Spring Harbor Laboratory Press, 1991.), develop improved agricultural stock (see, e.g., Ebert, K. M. et al., Animal Biotechnology 1:145-159 (1990)) and xenotransplantation (see, e.g., Osman, N., et al., Proc. natl. Acad. Sci USA 94:14677-14682 (1997)). However, the technique is limiting in that it only allows for the addition of genetic material to the developing embryo and not the deletion or modification of the endogenous genes. In addition, the microinjection of DNA into the nucleus is an inefficient process resulting in only 1-2% transgenic offspring from embryos injected and frequently producing mosaic animals which do not have the transgene in all cells.
The invention features a method of delivering DNA to a spermatogonium by infusing DNA in situ into a testicle of a non-human animal and administering a condition or substance to the testicle to increase uptake of DNA by the spermatogonium. By xe2x80x9cin situxe2x80x9d is meant in the original location in the body of the animal. Surgical exposure of the testis is not required. For example, a needle is inserted directly into testicular tissue and DNA delivered using a syringe. In some cases, the epididymis is surgically exposed at the head, and the DNA is delivered to the testes via a retrograde flush through the rete testes into the seminiferous tubules.
By xe2x80x9cspermatogoniumxe2x80x9d is meant an unspecialized diploid germ cell which can undergo mitosis and meiosis to give rise to a sperm cell. For example, a spermatogonium is a primordial germ cell which can differentiate into a sperm cell. Preferably, the spermatogonium is located on the walls of the basal membrane of the seminiferous tubule. To increase uptake of DNA by a spermatogonium, the testicle is exposed to a condition such as passage of an electrical current through the testicle. The electrical current is applied using a defibrillator or electroejaculator. Application of an electrical current is a means to electroporate the DNA into the target cells, i.e., spermatogonia. DNA uptake may also be enhanced by administering a substance such as a lipid or phospholipid. The condition or substance is administered to testicle either simultaneously with the infusion of DNA or after the DNA has been infused. DNA is administered as naked DNA or by viral infection, e.g., packaged into a viral vector such as adenovirus, adeno-associated virus. By xe2x80x9cnakedxe2x80x9d is meant free from any delivery vehicle that facilitates entry into the cell. For example, a naked DNA preparation is free of viral proteins or particles, DEAE-dextran, phospholipids, lipids, or calcium phosphate. The DNA contains a sequence encoding a selectable marker such as DNA encoding an antibiotic resistance gene, a cell surface antigen, or thymidine kinase. Following administration of the DNA, transformed or transfected cells are selected by administering an antibiotic, antibody-toxin complex, or chemical agent either systemically or locally to the testes to kill cells which do not express the DNA or to identify cells which express the DNA. The terms xe2x80x9ctransformxe2x80x9d and xe2x80x9ctransfectxe2x80x9d are used interchangeably throughout the specification; these terms refer to means of transferring or delivering DNA to a cell. A xe2x80x9ctransfectedxe2x80x9d or xe2x80x9ctransformedxe2x80x9d cell is one that contains the DNA sought to be delivered to it. The DNA may also contain a second promoter which directs expression of an apoptotic gene to selectively kill germ cells which have not undergone homologous recombination with the administered DNA The DNA is administered in a volume of solution sufficient to infuse the entire testicle, e.g., 0.1 ml per testicle/per treatment for a newborn animal and up to 5 ml per testicle/per treatment for an adult animal. Repeated treatments may be carried out if desired, e.g, to increase DNA uptake.
Preferably, the non-human animal is a sheep, goat, pig, cow, chicken, rabbit, rat, mouse, or guinea pig. More preferably, the animal is prepubetal, e.g., at an age at which the testicle has not yet begun to produce sperm. For example, the preferred age of a pig is at least 30 days but not greater than 100 days. At this age, the number of target cells, i.e., spermatogonia, is relatively low. An advantage of this approach is that destruction of spermatogenic cells prior to administration of DNA is not required.
Also within the invention is a method of making a non-human transgenic animal comprising by infusing DNA in situ into a testicle of a prepubetal non-human animal, harvesting sperm cells from the animal, contacting an ovum with said the cells under conditions suitable for fertilization, and producing a non-human transgenic animal. By xe2x80x9ctransgenic non-human animalxe2x80x9d is meant an animal that has gained (or lost) a DNA sequence from the introduction of an exogenous DNA sequence, i.e., transgene, into its own cells, or into an ancestor""s germ line. Such animals are produced by natural breeding, artificial insemination, or in vitro sperm injection into ova. By the term xe2x80x9ctransgenexe2x80x9d is meant any exogenous DNA sequence which is introduced into both the somatic and germ cells or only some of the somatic cells of a mammal. The transgene may or may not be an integral part of a chromosome. If the transgene is integrated into a chromosome, it may or may not be located at the same site as its corresponding endogenous gene sequence.
The transgene is preferably driven by a tissue specific promoter. For example, for protein expression in mammary glands, the human lactoferrin promoter is operably linked to DNA encoding a desired protein or polypeptide. Other promoters which direct preferential expression of a polypeptide in mammary gland tissue include the casein promoters (e.g., goat beta casein promoter and alpha S1 casein promoter) and whey protein promoters (e.g., whey acid protein promoter, beta lactoglobulin promoter, and alpha lactalbumin promoter). The promoter is inducible or constitutive.
Organs from transgenic animals produced according to the invention are useful for xenotransplantation. For example, the transgenic non-human animal the organs of which are suitable for transplantation into human recipients expresses human CD59 and/or lacks expression of porcine CD59 (a CD59 xe2x80x9cknockoutxe2x80x9d pig) or the animal expresses human H-transferase and/or lacks porcine Gal(xcex11,3) galactosyl transferase.
The methods of the invention solve many ongoing problems in DNA delivery for the generation of transgenic animals. For example, an adenovirus genome can only carry a transgene of limited size. The use of sperm does not limit the transgene size. For example, DNA as small as a few base pairs (bp) and as large as 100-200 kilobases (kb) are delivered using the methods of the invention. Typically, approximately 5 kb, 10 kb, 20 kb or 25 kb are delivered to target cells. However, up to 400 kb may be transferred to cells. For example, more than one vector or DNA fragment is transfected simultaneously to allow for the production of multimeric proteins (i.e., immunoglobulins, FAb fragments, fibrinogen, and collagen) or expression of more than one protein coding sequence.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.